This invention relates to a method of multi-color labeling at least two antigens being simultaneously present in a common biological system, with the aid of a corresponding number of different antibodies. In a second aspect the invention relates to a novel kit for carrying out the aforesaid method.
Fluorescene-optical multi-color labeling of different antigens in the same biological system, in particular animal, and especially human "tissue" (meaning non-liquid tissue as well as blood) is of special importance for investigating diagnostic or medical scientific problems.
For instance, such labeling is useful in the precise identification of lymphocyte subpopulations in pathologically and non-pathological tissues such as inflammatory myopathies, or auto-immune thyreoiditis, normal and pathologically changed lymphoid tissues, infiltrations or inflammatory changes in malignant and non-malignant tumors and metastases of malignant tumors, blood and cerebral fluid in pathological and non-pathological states diagnoses of immunodeficiency syndromes in blood such as AIDS and others listed on page 28 of a publication entitled "Ortho-mune (R), Monoklonale Antikorper, Zelltypisierung in Peripherblut and lymphatischem Gewebe" by Dr. Molter GmbH, D-6903 Neckergemund, Federal Republic of Germany, and many other fields of biological investigation.
Hitherto, it was only possible to effect multi-markings of blood, but not of other tissues with very costly and highly complicated laser analyzers such as a FACS analyzer or a Spectrum 3 analyzer which are available, due to their cost, in only a very limited number of medical institutions.
Methods published by William E. Gathings, Ph.D. and others in 1977 in the European Journal of Immunology 7, page 804 under the title of "Immunofluorescent Studies of Development of Pre-B Cells, B-Lymphocities and Immunoglobulin Isotype Diversity in Humans" have been reported by Becton Dickinson Labor Systems' "Monoclonal Antibody Source Book" published subsequently in Heidelberg in the Chapter "Methods--Immunofluorescence Staining of Cell Surfaces--Cytocentrifuge Preparations" comprise two-color indirect immunofluorescence preparations of human peripheral blood with the following reagents:
(1) Fluorescine(FITC) or rhodamine (RITC) labeled mouse monoclonal antibody specific for membrane antigen, as well as, for double-marking: PA0 (1a) unconjugated or biotin-labeled mouse monoclonal antibody specific for membrane antigen, followed by PA0 (1b) anti-mouse Ig FITC/RITC) or followed by PA0 (1c) Avidin FITC/RITC.
I have found that this method will not work for double-marking when (1a) is unconjugated and (1b) is anti-mouse Ig FITC/RITC, when the unconjugated antibody of (1a) is of the same immunoglobulin class as directly conjugated antibody of (1), as this method yields unspecific double markings.
Moreover, biotin-labeled mouse monoclonal antibody of (1a) followed by avidin FITC/RITC of (1b) as obtained from Becton and Dickinson fails to afford specific double markings.
However, even when using in the last combination an avidin FITC/RITC of different origin, I obtained only weak and quickly fading fluorescent markings. The weakness and fading were particularly pronounced in the green (FITC) markings.
Another method of fluorescence double marking of primary antibodies from the same animal species has been published by Amersham Buchler GmbH and Co. KG, D-3300 Braunschweig in "The biotin-streptavidin system" published in April 1985 (penultimate sheet). This method always leads to unspecific fluorescence double markings of the first incubation sequence.
Furthermore, D. Y. Mason, Z. Abdulazig, B. Falini and H. Stein assert on page 119 in an article entitled "Double Immunoenzymatic Labelling" published in "Immunocytochemistry, Practical Applications in Pathology and Biology", by G. Wright, Bristol and Boston, 1983 that it is "not possible to perform double labeling by" use of class- or subclass-specific antibodies (FIG.7.6) "if the two primary antibodies are of the same subclass. Since many monoclonal antibodies are of the Ig Gl subclass this represents an important potential limitation" of this known method.